Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/49—Blood
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

• the present invention relates to the detection of protein in aqueous fluids, e.g. urine, by the use of a test system containing a protein error indicator and a buffer.
• a test system containing a protein error indicator and a buffer.
• the determination of the presence of protein in a urine sample is important in the diagnosis of several pathological conditions affecting the kidney, circulatory and central nervous systems. It is often necessary to qualitatively and quantitatively measure protein in urine, especially in connection with the diagnosis of diabetes and kidney disease.
• the predominant urine protein associated with diabetes is albumin which is the protein most commonly sought out in analysis.
• Protein error indicators are pH indicators which contain an ionizable group which is displaced in the presence of protein to provide a detectable color change. This is the same color change that the indicator would undergo under the influence of a pH change, so it is important to include a buffer in the test system to thereby avoid a pH increase since such an increase could cause the color change in the indicator in the absence of protein thereby resulting in a false positive result.
• Protein detection methods based on the binding of protein error indicators such as phenolsulfonephthalein dyes are relatively nonspecific means of protein determination.
• the present invention involves the use of certain competitive inhibitors to increase the specificity of methods based on the binding of protein error indicators.
• United States patent 5,187,104 discusses the use of DIDNT dye in a protein detection method and mentions the use of color enhancing polymers in combination with the reagents.
• Specific polymers mentioned are polypropylene glycols, poly(propylene ether carbonate), and polyvinylethers.
• polyether carbonate designated as KOK 10,002 from Bayer AG, a propylene oxide and ethylene oxide adduct of 1,6-dimethyl-4-nonylphenol available from Bayer AG under the tradename Fenoil D4030 and a polyvinyl ether available under the designation Lutonal ISO from BASF.
• polyvinyl alcohol has been described in conjunction with protein tests based on metal chelating dyes by Y. Fujiti in Bunseki (32) 379-386 (1983).
• This reference describes polyvinyl alcohol and polyvinyl pyrrolidone as suitable nonionic surfactants for unicell formation but does not mention any increase in the specificity for albumin.
• the present invention involves the semi-quantitative analysis of human serum albumin in an aqueous test sample which is carried out by contacting the fluid suspected of containing this protein with a test reagent comprising a protein error indicator dye which undergoes a detectable color change when contacted with protein.
• a competitive inhibitor characterized by formula A: wherein is a linking group in which A and B can be a single bond or oxygen so that when A is oxygen and B is a bond the polymer is a poly(vinyl alkyl ester), when A is a bond and B is oxygen, the polymer is a poly(alkyl acrylate), when A is oxygen and B is oxygen, the polymer is a carbonate and when A is a bond and B is a bond the polymer is a poly(vinyl alkyl ketone).
• the R substituent is a straight, branched or cyclic alkyl group of 1 to 20 carbon atoms in which 0 to 10 hydrogens can be replaced by hydroxyl groups
• the polymer backbone comprises repeating alkyl or carbonate, e.g. cellulose or glucoamine, sub-units which can be co-polymerized with unreactive blocking units and the number of alkyl groups attached to repeating polymer sub-units through the linking groups ranges from 10% to 90% of the theoretical maximum.
• polymeric inhibitors reduce the response of a protein error indicator to proteins in the protein error indicator test under consideration.
• Those particular polymers that inhibit the protein error indicator response to human serum albumin (HSA) to a lesser degree than the response to other proteins normally found in urine are desirable additives to a reagent for determining the concentration of HSA, because of the resulting increase in specificity for HSA which such a system provides.
• Those polymeric inhibitors which are useful in the present invention are represented by formula A.
• the polymers represented by this formula can be poly(vinyl alkyl esters), poly(alkyl acrylates), poly(vinyl alkyl carbonates) or poly(vinyl alkyl ketones).
• poly(vinyl alkyl esters) i.e. polymers in which A is oxygen and B is a bond in the foregoing formula
• specific polymers in addition to those set out in Table 5 include, but are not limited to cases where the alkyl groups are, cyclohexenoate, trans-5-decanoate, 10-hydroxydecanoate and 5-hydroxydecanoate.
• a poly(alkyl acrylate) is used, i.e.
• suitable polymers in addition to those listed in Table 5 include those in which the alkyl groups are octyl, cyclohexyl, dicyclohexyl and 5-methylhexyl.
• polymers according to the foregoing formula in which both A and B are oxygen in addition to those set out in Table 5 include those in which the alkyl groups are nonadecyl, decyl, 4-hydroxyheptyl and cyclopentadiene, whereas poly(vinyl ketones) which are represented when both A and B are bonds include alkyl groups such as butyl, decyl, neodecanoate and octadecyl as well as those in Table 5.
• the molecular weights of these polymers are not critical to their usefulness in the present invention although those in which the degree of polymerisation (n) ranges from about 20 to 40,000 are preferred due to their ability to adhere to the surface of the HSA molecule.
• One aspect of the present invention is directed to an analytical test strip for the detection of HSA in urine which strip comprises an absorbant carrier impregnated with a suitable protein error indicator, a suitable buffer and the polymeric inhibitor.
• suitable protein error indicators include Tetrabromophenol Blue (TBPB), 5',5''-Dinitro-3'3''-Diiodo-3,4,5,6-Tetrabromophenolsulfonephthalein (DIDNTB), Coomassie Brilliant Blue, Fast Green FCF, Light Green SF, pyrogallol red and pyrocatechol violet.
• TBPB Tetrabromophenol Blue
• DIDNTB 5',5''-Dinitro-3'3''-Diiodo-3,4,5,6-Tetrabromophenolsulfonephthalein
• DIDNTB 5',5''-Dinitro-3'3''-Diiodo-3,4,5,6-Tetrabromophenolsulfonephthalein
• the absorbant carrier of the test strip is preferably filter paper.
• Other materials useful as the absorbant carrier include felt, porous ceramic strips and woven or matted glass fibers such as those described in U.S. Patent 3,846,247. Also suitable are wood, cloth, sponge material and argillaceous substances such as those described in U.S. Patent 3,552,928.
• the absorbant carrier can be of a nonporous material such as a polymeric film or glass.
• the absorbant carrier is impregnated with a solution of the protein error indicator, buffer and the polymeric inhibitor.
• This impregnation is normally carried out by a two dip procedure in which the first dip comprises water or a water/polar organic solvent mixture in which there is dissolved a buffer.
• the strip is dipped into a second solution of an organic solvent in which is dissolved the protein error indicator which is typically present at a concentration of from about 0.2 to 5.0 mM and the polymeric inhibitor.
• the strips After dipping and drying, the strips are ready for use which normally involves dipping them into a urine sample and reading the response resulting from the color change in the indicator which reading is conducted either manually or by use of a reflectance spectrometer for better quantitation.
• the pH at which the assay is conducted will depend on the particular dye which is used in the reagent formulation.
• the buffers which are most compatible with a particular dye are known or can be readily determined through routine experimentation.
• the method of practicing the present invention is further illustrated by the following examples. These examples and the data contained therein demonstrate the desirability of using polymers, as described by the foregoing general structure, to increase the specificity of the dye binding method for human serum albumin (HSA). This method enhances the diagnostic value of urinary HSA determinations and allows more accurate assessments of kidney health.
• HSA human serum albumin
• the HSA specificity of a phenolsulfonephthalein dye [5',5''-dinitro-3',3''-diiodo-3,4,5,6-tetrabromophenolsulfonephthalein (DIDNTB)] was measured in the presence of increasing concentrations of several types of polymeric and non-polymeric additives. Also tested were certain long chain substituted alkanes. The general structure of the additives tested is set out in the following Scheme I.
• the DIDNTB protein reagent was made from two saturations of Alhstrom 204 filter paper in which the first saturation was made with an aqueous ethanol mix containing tartaric acid as buffer and methyl red as background dye. The pH was adjusted to 2.1. The second saturation was with a toluene/tetrahydrofuran mix containing the DIDNTB indicator dye and Lutanol M40 (a polyvinylmethylether) as a color enhancer polymer.
• Water soluble additives such as poly(vinyl alcohol), poly(vinylsulfonic acid) and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) were added to the first mix while water insoluble additives such as decanol, decanoic acid, hexadecanoic acid, poly(vinyl stearate), poly(styrene/maleic anhydride) and poly(sodium-4-styrene sulfonate) were added to the second mix.
• water insoluble additives such as poly(vinyl alcohol), poly(vinylsulfonic acid) and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) were added to the first mix while water insoluble additives such as decanol, decanoic acid, hexadecanoic acid, poly(vinyl stearate), poly(styrene/maleic anhydride) and poly(sodium-4-styrene s
• the mix solutions were used to saturate the filter paper and the paper was dried at 105°C for 7 minutes after each saturation.
• the resultant dry reagents were processed into reagent strips which were tested on a Clinitek-200+ reflectance spectrophotometer from Bayer Diagnostics after dipping into urine containing 0 or 30 mg/dL human serum albumin or 40 mg/dL of another urinary protein.
• the particulars of each dip solution are set out in Table 3.
• the urine sample Prior to the addition of HSA, the urine sample was filtered through an ultrafiltration membrane having a 10 KDa (kilodalton) molecular weight cut-off to remove naturally occurring proteins.
• the total protein in the urine sample was determined using an immunological HSA assay and the Coomassie Brilliant Blue (CBB) method described by Perini et al as cited above to screen over 175 clinical samples to provide specimens lacking albumin but containing other urine protein.
• CBB Coomassie Brilliant Blue
• the reagent response was measured on the CLINITEK-200+ instrument as the result of 1000 x % Reflectance @ 610 nm/% reflectance @ 690 nm. The difference between negative and protein containing urines was taken as the protein response.
• the data generated by this experiment are set out in Table 1 in which the protein response of control formula lacking polymer or substituted alkane additive is compared to the formulas containing polymer or alkane to determine the % change in response. Negative numbers indicate greater loss of protein response.
• the HSA response in the presence of these additives was compared to the response for other urinary proteins such as Tamm Horsefall glycoprotein, Bence-Jones protein, ⁇ -1-microglobin, hemoglobin and various low molecular weight protein fragments.
• the additives were used at a 0.1% (w/w) concentration to allow a measurable HSA response in all cases.
• the data generated are set out in Table 2.
• the 0.1% concentration amounts to 10 to 20 ⁇ M concentration of additive and is in excess of the 4.4 ⁇ M concentration of the proteins tested. Only poly(vinyl stearate) inhibited other urinary protein more than it inhibited HSA indicating that this polymer increases the specificity of the phenolsulfonephthalein method for albumin.
• poly(vinyl stearate) increases the specificity of the phenolsulfonephthalein method for determining HSA in urine.
• Other additives either had no effect, being within 10% of control, or inhibited HSA and other urinary proteins equally as can be determined from Table 1. Since the presence of 6 mM of substituted alkanes such as decanol, decanoic acid and hexadecanoic acid did not affect the protein response, it can be determined that the effect of poly(vinyl stearate) cannot be attributed to the alkyl group of the polymer alone.
• DIDNTB dye was measured in the presence and absence of a series of alkyl polymers as set out in Scheme II.
• DIDNTB dye was assessed with other polymers typically used in protein reagents. These polymers were Lutanol M40 (described in U.S. 5,424.215) KOK 10071 polymer (described in U.S. 5,424,215) and poly(vinyl alcohol). These polymers did not increase the specificity of DIDNTB dye for albumin. The KOK 10071 and Lutanol M40 polymers increased the sensitivity of the test for all proteins whereas PVA had no significant effect. This was expected based on the teachings of U.S. Patents 5,187,104 and 5,424,125. The data generated in comparing these polymers are set out in Table 4. Comparison of Typical Polymers.
• HSA albumin
• Hb hemoglobin
• TRANS transferrin
• LYS lysozyme
• Esters, acrylates, ketones and carbonates all improved specificity as shown by a decreased lysozyme sensitivity with either a constant or less reduced HSA sensitivity. The best is observed with hexanoate, neodecanoate, decyl and decanoate alkyl groups. Polymers with these groups also reduced the hemoglobin and transferrin sensitivity. Branched and hydroxyl containing alkyl groups and a carbohydrate polymer backbone with an ester alkyl group were also effective.
• Example II The experimental procedure for Example II involved two saturations from filter paper as in the previous example.
• the first saturation was with an aqueous ethanol mix containing a citric acid buffer and PVA as a binding polymer and the mix pH was adjusted to 2.1.
• the second saturation was a toluene mix containing a protein indicator dye (DIDNTB) and enhancer polymers (Lutanol M40 and KOK 10071).
• the mix solutions were used to saturate filter paper whereupon the paper was dried at 105°C for 7 minutes after each saturation.
• the function, preferred concentration and allowable range of ingredients in the reagent formulation are set out in Table 6.
• Ingredient Function Conc Ingredient Function Conc.

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• 1996
  • 1996-03-01 US US08/609,674 patent/US5750405A/en not_active Expired - Lifetime
  • 1996-09-04 TW TW085110807A patent/TW438976B/zh active
  • 1996-11-04 CA CA002189586A patent/CA2189586A1/en not_active Abandoned
• 1997
  • 1997-02-18 EP EP97102554A patent/EP0793099A1/de not_active Withdrawn
  • 1997-02-26 CN CN97102543A patent/CN1165301A/zh active Pending
  • 1997-02-27 KR KR1019970006081A patent/KR970065286A/ko not_active Ceased
  • 1997-02-27 JP JP9043318A patent/JPH09243638A/ja active Pending
  • 1997-02-28 AU AU15016/97A patent/AU706440B2/en not_active Ceased

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